Bacterial biofilms, the structures created by surface attached bacteria, are the basis of infections that are recalcitrant to antibiotics. For Vibrio cholerae, the Gram negative bacterium that causes the diarrheal disease cholera, these structures facilitate attachment to environmental surfaces. An understanding of the process by which these structures form is essential to blocking the process. V. cholerae responds to specific environmental conditions by synthesizing an adhesive extracellular matrix that promotes biofilm formation. This matrix is comprised of the VPS exopolysaccharide, proteins, and DNA. Through a proteomic analysis of this matrix, we recently identified three secreted proteins, Bap1, RbmC, and RbmA, that are required for the structural integrity of the V. cholerae biofilm. Bap1 and RbmC, which are concentrated between the biofilm and the substratum, mediate adherence of the biofilm structure to the surface. In contrast, RbmA is dispersed throughout the biofilm and surrounds biofilm-associated cells. Based on structural data, RbmA is hypothesized to bind both the bacterial O-antigen and VPS polysaccharide, pulling the biofilm matrix onto the bacterial cell surface. During our studies, we noted that RbmA undergoes proteolytic cleavage in mature biofilms. Our preliminary results suggest that premature cleavage of RbmA augments recruitment of cells to the biofilm. In this application, we propose to evaluate a model in which cleavage increases the affinity of RbmA both for the O-antigen and the VPS polysaccharides, thus locking in the biofilm structure once cell growth within the biofilm is complete. We will tet this model by identifying the RbmA protease or proteases, elucidating the mechanisms by which proteolysis of RbmA is regulated, and exploring the impact of RbmA proteolysis on RbmA function and resistance of mature biofilms to mechanical stress. These studies will define a new paradigm for the role of proteolysis in bacterial biofilm maturation and may suggest new technologies to prevent reinforcement of the biofilm matrix during this process.